IL180635A - Use of an injectable methylene blue dye for sentinel lymph node identification - Google Patents

Use of an injectable methylene blue dye for sentinel lymph node identification

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IL180635A
IL180635A IL180635A IL18063507A IL180635A IL 180635 A IL180635 A IL 180635A IL 180635 A IL180635 A IL 180635A IL 18063507 A IL18063507 A IL 18063507A IL 180635 A IL180635 A IL 180635A
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cancer
dye
sentinel lymph
methylene blue
lymph nodes
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IL180635A
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Eugene A Woltering
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Univ Louisiana State
Eugene A Woltering
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/001Preparation for luminescence or biological staining
    • A61K49/006Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04Organic compounds

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Description

180635/2 180635 |7'ji I 453472 ΓΛΊΝ USE OF AN INJECTABLE METHYLENE BLUE DYE FOR SENTINEL LYMPH NODE IDENTIFICATION TECHNICAL FIELD
[0003] This invention pertains to a method to identify sentinel lymph node(s), node(s) that receive lymphatic fluid from a tumor tissue, by injecting into or around a tumor tissue during surgery a single active compound, a radiolabeled dye, that rapidly travels to the sentinel lymph node(s).
BACKGROUND ART
[0004] One of the major techniques for determining the prognosis of a cancer involves determining whether the cancer has metastasized into other areas of the body. As lymphatic fluid flows from various areas of the body, the fluid flows through lymph channels and then into lymph nodes where it is filtered. The initial spread of most solid tumors occurs as metastasizing cells move into the lymph channels, and then are filtered by the lymph nodes. The first lymph node that is reached by the lymphatic fluid from the tumor region is called the sentinel lymph node ("SLN")- See TJ. Miner et al, "Guidelines for the safe use of radioactive materials during localization and resection of the sentinel lymph node," Ann. Surg. Oncol., vol. 6, pp. 75-82 (1999). For example, the sentinel lymph nodes for breast tumors are usually found in the axilla, or armpit, of the patient. A tumor may have one or more sentinel lymph nodes.
[0005] A sentinel lymph node biopsy is used to determine whether all lymph nodes in the drainage area must be removed or only the SLN(s) are removed. This procedure depends upon an effective technique for identifying the sentinel lymph node(s) for a tumor. If the cancer has spread to the lymph nodes, the sentinel lymph node should be positive (i.e., cancerous), and the surgeon will then remove all lymph nodes in the region. If the SLN is pathologically negative, all other nodes of the same area are, generally cancer-free. See N. Cascinelli et al, "Sentinel lymph node biopsy in cutaneous melanoma: The WHO Melanoma Program Experience," Ann. Surg. Oncol., vol. 7, pp. 469-474 (2000). Thus, if the SLN is pathologically negative, the patient has lost only the SNL. Accurate identification and biopsy of the sentinel lymph node (when pathologically negative) means that other nodes (non-SLNs) are retained by the patient. The retention of normal lymph nodes benefits the patient by preventing lymphatic fluid accumulation in the arm or other extremity.
[0006] The current techniques for identifying the sentinel lymph node(s) involve the use of a radioactive colloid compound, a vital dye, or both.; For example, a blue dye is injected around the tumor during surgery and visually, stains the sentinel node(s) within 5 to 10 min. See International Patent Application No. 2004/016154. The' blue dye conventionally used for this sentinel lymph node procedure is an isosulfan blue dye (e.g., Lymphazurin™). A radioactive colloid, if used, is usually injected 2 to 24 hr before surgery. See G.F. Schwartz et al. , "Proceedings of the consensus conference on the role of sentinel lymph node biopsy in carcinoma of the breast, April 19-22, 2001, Philadelphia, PA, USA," The Breast Journal, vol. 8, pp. 126-138 (2002). The radiolabeled colloid enables the site of the sentinel node(s) to be remotely detected both by preoperative lymphoscintigraphy and by intraoperative handheld gamma probe detection. The radioactive colloid compound is usually a technetium-sulfur colloid (e.g., 99mTc-sulfur colloid, Mallinckrodt, St. Louis, Missouri). Unfiltered 99mTc-sulfur colloid is often used because its large particle size slows migration, and makes it less likely that the radioactive material will migrate through the initial sentinel lymph node and into second and third echelon nodes before detection. A particle size of 5 nm has been reported as optimal for the radioactive compound. Unfiltered 99mTc-sulfur colloid has been reported not to pass beyond the sentinel lymph nodes for at least the first six hours after injection. See F.L. Moffat, Jr., et al, "Unfiltered sulfur colloid and sentinel node biopsy for breast cancer: Technical and kinetic considerations," Ann. Surg. Oncol., vol. 6, pp. 746-755 (1999); and International Application No. WO 00/74727.
[0007] When both a dye and a radiolabeled substance are used, they have been injected separately, the radiolabeled substance before the surgery and the dye during surgery. Both substances are usually injected in or near the rumor or tumor site and flow through the lymphatic channels to the sentinel lymph node draining that site. The SLN can be located ^preoperatively by lymphoscintigraphy, and during surgery both by detecting the emissions from the radioactive substance and by visual inspection of nodes that are colored by the dye. See Cascinelli, 2000; and K.M. McMasters et al., "Sentinel lymph node biopsy for melanoma: How many radioactive nodes should be removed?" Ann. Surg. Oncol., vol. 8, pp. 192-197 (2001). Literally hundreds of studies have appeared , in the scientific literature validating the use of sentinel lymph node biopsy as an accurate method to evaluate the risk of metastatic disease in auxiliary nodes. Sentinel lymph node biopsy has been effectively used or proposed in several solid tumor types, including melanoma, breast cancer, head and neck cancer, lung cancer, bladder cancer, neuroendocrine cancer, squamous carcinoma, prostate cancer, gastric cancer, cervical cancer, vulvar cancer, thyroid cancer, pancreatic cancer, head and neck cancer, renal cancer, esophageal cancer, rectal cancer, penile cancer, lymphoma, multiple myloma, Merkel cell tumors, ovarian cancer, and colorectal cancer. See M.L. Echt et al., "Detection of sentinel lymph nodes with lymphazurin in cervical, uterine, and vulvar malignancies," Southern Medical Journal, vol. 92, pp. 204-208 (1999); H. Hayashi et al., "Sentinel lymph node mapping for gastric cancer using a dual procedure with dye- and gamma probe-guided techniques," J. Am. Coll. Surg., vol. 196, pp. 68-74 (2003); S. Saha et al., "Lymphazurin 1% versus 99mTc sulfur colloid for lymphatic mapping in colorectal tumors: a comparative analysis," Ami. Surg. Oncol., vol. 11, pp. 21-26 (2004); and International Publication No. WO 2004/016154. These reports document the success of sentinel node localization, usually using isosulfan blue dye, with or without also using a technetium 99-labeled sulfur colloid. Later reports indicate an improved accuracy at identifying the sentinel lymph node by using both dye and radioactive colloid. See Schwartz et al., 2002.
[0008] Recently several reports have appeared suggesting that sentinel node identification accuracy and yield could be duplicated with the use of methylene blue dye (3,7-bis (dimethylamino)phenothiazine-5-ium chloride) instead of isosulfan blue dye. See R. Simmons et al., "Methylene blue dye as an alternative to isosulfan blue dye for sentinel lymph node localization," Ann. Surg. Oncol., vol. 10, pp. 242-247 (2003). A change in dye preference has accordingly also found its way into the practice of many surgeons since methylene blue is less expensive and more readily available than the commonly used isosulfan blue dye, Lymphazurin™. Small quantities (usually about 4 to 5 cc) of methylene blue are injected into the tumor region. Methylene blue has not been associated with reported adverse events.
[0009] Sentinel node biopsy using radioactive colloid has some unanticipated consequences for both surgeon and patient. Patients usually undergo a separate procedure for Γ■ injecting the radioactive colloid prior to cancer surgery. This procedure is carried out either the afternoon prior to surgery or the morning of surgery. The patient can then be screened before the surgery by lymphoscintigraphy. The injection of radioactive colloid is unusually painful, whether it is injected in small quantities intradermally or in larger quantities around the tumor. With the increasing demand for sentinel lymph node sampling, surgeons have been forced to deal with major delays in surgical schedules. Although some surgeons have injected the radioactive colloid at the time of surgery to avoid preoperative lymphoscintigraphy, they must still wait 30 to 60 min for the radioactive colloid to reach the lymph nodes. See T.M. Turtle, "Technical advances in sentinel lymph node biopsy for breast cancer," Am. Surg., vol. 70, pp. 407-413 (2004); and International Application No. WO 00/74727.
[0010] Other shortcomings of the known procedures include a relatively insensitive degree of localization, i.e., the inability to discriminate nodes on a fine scale. 99mTc has high-energy gamma emissions. A significant amount of activity (10 mCi) must be injected to ensure adequate node uptake. A large compound, e.g., colloid, is used to slow loss of radioactivity from the lymph nodes. However, some of the activity must clear from the injection site (especially ones close to the axilla) before using a handheld gamma probe in the 180635/2 axilla, or the gamma detector will be "swamped," making it difficulty to distinguish a small radioactive sentinel node from a non-radioactive neighboring node. [001 1] Radioactive iodine-labeled methylene blue has previously been used for the early diagnosis and treatment of melanoma metastases. Methylene blue is used because it possesses a high affinity for melanin and accumulates preferentially in melanoma cells with high concentrations of melanin. See A. Raffaelli et al., "Investigation on the iodinatibn reaction of methylene blue by liquid chromatography-mass spectrometry with ionspray ionization," Journal of Chromatography A, vol. 854, pp. 57-67 (1999); E.M. Link, "Targeting melanoma with 21 lAt/131I-methylene blue: preclinical and clinical experience," Hybridoma, vol. 18, pp. 77-82 (1999); E.M. Link et al., "Early detection of melanoma metastases with radioiodinated methylene blue," Eur. J. Nucl. Med., vol. 25, pp. 1322-1329 (1998); and E.M. Link et al., "Uptake and therapeutic effectiveness of l25I- and 2 , 1At-methylene blue for pigmented melanoma in an animal model system," Cancer Res., vol. 49, pp. 4332-7 ( 1989). Early research on use of I-labeled methylene blue to image and localize parathyroid adenomas proved unsatisfactory. See PJ. Blower et al., "123I-methylene blue: an unsatisfactory parathyroid imaging agent," Nuclear Medicine Comms., vol. 13, pp. 522-527 (1992).
[0012] International Publication No. WO 2004/016154 discloses the use of a marker nucleic acid segment to aid in sentinel lymph node biopsy.
[0013] International Publication No. WO 00/74727 discloses the co-injection of a visible blue dye with a radiopharmaceutical agent (comprising a probe with a radiolabel) for lymphoscintigraphy. It discloses linking the dye to a probe that is radiolabeled, e.g., isothiocyanate to 99mTc sulfur colloid, and the importance of size of the radiopharmaceutical agent. The list of probes include molecules of large molecular weight (e.g., colloids, starch, dextran, albumin, etc.) to achieve an optimal size of 5 run. The minimum detection time after injection was 30 min.
DISCLOSURE OF INVENTION In one embodiment, the present invention provides the use of radiolabeled methylene blue in the manufacture of an injectable medicament for identifying, in a mammalian patient with a solid tumor, one or more sentinel lymph nodes to which lymph from the tumor initially flows; wherein said injectable medicament is suitable for a method comprising the steps of: (a) injecting radiolabeled methylene blue in or near the tumor or tumor site; (b) identifying as a sentinel lymph node a lymph node that, within about 20 minutes following said injecting, has 180635/1 accumulated a detectable fraction of the radiolabeled methylene blue, as indicated optically by the presence of the dye methylene blue, or as indicated radiologically by decay products of the radiolabel, or both; and (c) surgically removing the identified one or more sentinel lymph nodes.
In certain embodiments, the identifying step in a use of the present invention comprises identifying the location of one or more sentinel lymph nodes radiologically during surgery; and then further identifying the location of the one or more sentinel lymph nodes by optically locating dyed tissue during surgery, in one or more locations that had thus been indicated radiologically; and said method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
In other embodiments, the radiolabled methylene blue that is utilized is present in said injectable medicament as an admixture with a non-radiolabeled dye; the presence of the non-radiolabeled dye in a tissue may be observed optically due to the non-radiolabeled dye's visible, ultraviolet, or infrared absorption, fluorescence, luminescence, or phosphorescence; and the molecular weight and chemical properties of the non-radiolabeled dye are such that a detectable fraction of the non-radiolabeled dye will, following injection, travel from the tumor or the vicinity of the tumor to one or more sentinel lymph nodes within about 20 minutes. Several preferred embodiments of this method will now be described: In certain preferred embodiments, the identifying step comprises identifying the location of one or more sentinel lymph nodes radiologically during surgery; and the method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
In certain other preferred embodiments, the identifying step comprises identifying the location of one or more sentinel lymph nodes by optically locating dyed tissue during surgery; and the method additionally comprises the step, following said identifying, of surgically removing the .identified one or more sentinel lymph nodes.
In certain other preferred embodiments, the identifying step comprises identifying the location of one or more sentinel lymph nodes radiologically during surgery; and then further identifying the location of the one or more sentinel lymph nodes by optically locating dyed tissue during surgery, in one or more locations that had thus been indicated radiologically; 5A 180635/1 and the method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
In certain other preferred embodiments, the non-radiolabeled dye that is utilized is methylene blue.
In certain other preferred embodiments, the ratio of the non-radiolabeled dye to the radiolabled methylene blue is at least about 10: 1.
In certain other preferred embodiments, the molecular weight and chemical properties of the non-radiolabeled dye that is utilized are such that a detectable fraction of the dye will, following injection, travel from the injection site to one or more sentinel lymph nodes within about 15 minutes.
In certain other preferred embodiments, the molecular weight and chemical properties of the non-radiolabeled dye that is utilized are such that a detectable fraction of the dye will, following injection, travel from the injection site to one or more sentinel lymph nodes within about 10 minutes.
In certain other preferred embodiments, the non-radiolabeled dye that is utilized is selected from the group consisting of patent blue dye, isosulfan blue, methylene blue, fluorescein, tartrazine, iodocyanine green, rose Bengal, and congo red.
In certain other preferred embodiments, the radiolabled methylene blue is 125I-methylene blue and the non-radiolabeled dye is isosulfan blue.
In other embodiments of the present invention, the radiolabled methylene blue that is utilized is 125I-methylene blue.
In still other embodiments of the present invention, the target tumor is selected from the group consisting of melanoma, breast cancer, head and neck cancer, lung cancer, bladder cancer, neuroendocrine cancer, squamous carcinoma, prostate cancer, gastric cancer, cervical cancer, vulvar cancer, thyroid cancer, pancreatic cancer, renal cancer, esophageal cancer, rectal cancer, penile cancer, lymphoma, multiple myloma, Merkel cell tumors, ovarian cancer, and colorectal cancer. 5B 180635/2 In other embodiments of the present invention, the radiolabel that is utilized emits particles with an energy of less than about 300 keV. In other embodiments, the energy is less than about 150 keV. In other embodiments, the radiolabel energy is less than about 50 keV.
In other embodiments of the present invention, the radiolabel that is utilized is an isotope of an element selected from the group consisting of Tc, Re, Mn, Fe, Se, Co, Ni, Zn, Cd, Mo, W, Cu, Ag, Au, Tl, Hg, Cr, Rh, B, I, CI, F, At, Y, Lu, Gd, Ga, Ho, In, Sm and Yb. In other embodiments, the radiolabel is selected from the group consisting of l25l, 11 'in, 75Se, 57Co, l2 I, 90Y, 68Ga, 123I, 1241, 18F, ,3 ,I, "mTc, l69Yb, 186Re, 188Re, 2UAt, 77Br, 67Ga, 86Y, 193Pt, ,95mPt, and BRIEF DESCRIPTION OF DRAWINGS
[0015] Fig. 1 illustrates the radioactivity measured in the right paw at several time periods after injection of 125I-methylene blue for each of three rabbits, and an average of the three rabbits.
[0016] Fig. 2 illustrates the radioactivity measured in vivo in the sentinel lymph node at several time periods after injection of 125I-methylene blue for each of three rabbits, and an average of the three rabbits.
[0017] Fig. 3 illustrates the radioactivity measured in vitro in the sentinel lymph node at several time periods after injection of 125I-methylene blue for each of three rabbits, and an average of the three rabbits.
[0018] Fig. 4 illustrates the radioactivity measured in the neck (the background radioactivity) at several time periods after injection of 125I-methylene blue for each of three rabbits, and an average of the three rabbits. 6 180635/2
[0019] Fig. 5 illustrates the radioactivity measured in the left paw at several time periods after injection of 125I-methylene blue for each of three rabbits, and an average of the three rabbits.
[0020] Fig. 6 illustrates the radioactivity measured in blood (0.1 ml) at several time 1 7^ periods after injection of I-methylene blue for each of three rabbits, and an average of the three rabbits.
[0021] Fig. 7 illustrates the radioactivity measured in several organs at several time periods after injection of 125I-methylene blue for one rabbit at each time period.
[0022] Fig. 8 illustrates the radioactivity measured in the right groin region at several time periods after injection of I25I-methylene blue for each of three rabbits and an average of the three rabbits.
Any sections of the specification that fall outside the scope of the claims are not part of the present invention.
[0023] Several dyes may be used to visualize the lymph nodes. The dye may, for example, be a nonfluorescent dye, a fluorescent dye, an ultraviolet fluorescent dye, a visible fluorescent dye, and infrared fluorescent dye, a chemiluminescent dye, a phosphorescent dye, or a bioluminescent dye. The dye preferably has the following properties: (1) a small molecule (molecular weight less than about 2000) that is nontoxic, non-pyrogenic, and that can be sterilized; (2) a molecule that can be either radiolabeled directly or chelated to a radioisotope; and (3) a molecule that will be transported rapidly through the lymph channels to the lymph node(s). Examples of low molecular weight dyes that are useful in this invention include isosulfan blue (molecular weight, 566.70), patent blue dye (e.g., Patent Blue-V), methylene blue (molecular weight, 319.85), tartrazine (molecular weight, 534.39), iodocyanine green (molecular weight, 774.99), rose Bengal (molecular weight, 1049.84), congo red (molecular weight, 696.76), fluorescein (molecular weight, 332.30) and their respective derivatives. A list of potential dyes useful for this technology is disclosed for example in International Publication Nos. WO 00/74727 and WO 2004/016154, the disclosures of both of which are expressly incorporated by reference. The size of 7 180635/1 the radiolabeled dye should be small enough that the compound travels from the site of injection near the tumor to the sentinel lymph node in less than about 25 min, and most preferably in less than about 20 min. This small size requirement for the radiolabeled compound is in stark 7A contrast to prior references that recommend an optimal particle size of about 5 nm, specifically to slow the migration of the radiolabeled colloid compound.
[0024] Examples of radiolabels that are useful in this invention include radioisotopes that can be directly attached to the dye or chelated to the dye molecule and that can be detected using a handheld probe during surgery. Isotopes that emit gamma or x-rays are especially useful, although radioisotopes emitting beta particles (electrons or positrons), or alpha particles could be useful depending on the type of probe available. Examples of elements whose radioisotopes might be used include, but is not limited to, the following: Tc, Re, Mn, Fe, Co, Ni, Zn, Cd, Se, Mo,~W, Cu, Ag, Au, Tl, Hg, Cr, Rh, B, I, CI, F, At, Y, Lu, Gd, Ga, Ho, In, Sm and Yb. In addition, the most preferred isotope is one with detectable radiation that readily can be specifically localized by a radiation detection probe placed adjacent to the lymph node. As specific localization is the goal, the preferred isotopes are those that decay via small energy transitions (30 to 300 keV) and have highly localized dose profiles. For gamma and x-ray emitters, preferred isotopes emit photons with energies less than about 300 keV, more preferred less than about 150 keV, and most preferred less than about 50 keV, e.g., 12SI. Other low energy isotopes include 11 'in, 75Se, and 57Co. See U.S. Patent No. 4,782,840. Radioisotopes emitting beta particles (e.g., 124I, 90Y, 18F, and 68Ga) additionally can be used in conjunction with a probe that can detect either beta electrons or positrons. The detection of beta radiation intraoperatively is disclosed, for example, in U.S. Patent No: 5,008,546, the disclosure of which is incorporated herein by reference. Isotopes that emit higher energy particles (e.g., 131I) may also be used, although suitable collimation of the radiation detection probe must be employed, which may impede the instrument being facile to the surgeon and may limit the areas within the body cavity that can be suitably surveyed. Other isotopes that might be used include 123I, 99mTc, 169Yb, 186Re, 188Re, 21 'At, 77Br, 67Ga, 86Y, 193Pt, 195mPt, and 201T1. The optimal combination of isotope and probe will allow localization of a node with a resolution less than 1 cm, preferably less than 1 mm.
[0025] The ratio of radiolabeled dye to unlabeled dye depends on the isotope and sensitivity of the probe. The total amount of dye, both labeled and unlabeled, must be sufficient to visually see the lymph channels and lymph nodes. The amount usually recommended is about 4 cc to about 5 cc. The amount of the radiolabeled dye should be 8 sufficient that a SNL will have enough radioactivity to be detected by a hand held probe to be clearly distinct from background radioactivity, an amount from about 300 to about 2000 μθϊ, more preferably about 400 to about 1500 μθί, and most preferably about 1000 μθ.
[0026] The radiolabeled dye can be used to identify sentinel lymph node(s) of any solid tumor that is known to metastasize, including melanoma, breast cancer, head and neck cancer, lung cancer, bladder cancer, neuroendocrine cancer, squamous carcinoma, prostate cancer, gastric cancer, cervical cancer, vulvar cancer, thyroid cancer, pancreatic cancer, head and neck cancer, renal cancer, esophageal cancer, rectal cancer, penile cancer, lymphoma, multiple myloma, Merkel cell tumors, ovarian cancer, and colorectal cancer.
MODES FOR CARRYING OUT TEE INVENTION EXAMPLE 1 Time-Line and Distribution of Radiolabeled Methylene Blue
[0027] To determine the time line andt distribution for a radiolabeled dye, twenty-four rabbits of mixed ages and sex were injected at the same time. The animals were injected with 1 ml 125I-methylene blue (1 mCi/ml; l25I-3,7-bisdimetliylaminophenazathionine; ISO-Tex Diagnostics, Inc., Friendswood, Texas), a radiolabeled iodine salt of methylene blue, in the right hind paw. Groups of three animals were sacrificed at eight time periods: 0.25 hr, 0.5 hr, 1.25 hr, 2 hr, 4 hr, 8 hr, 24 hr, and 48 In. At each of these times the rabbits underwent dissection by three researchers working in parallel. Additionally, radioactivity in the lymph nodes was monitored using two different Neoprobe model 1000 handheld gamma probes. The rabbits were dissected to find the popliteal fossa, the nodes that were radioactive emitters, and the nodes with blue coloration. The overall biodistribution of radioactivity was determined by monitoring the following tissues: heart, liver, lung, stomach, intestine, kidneys-adrenals (combined), spleen, testes or ovaries, thyroid, overall carcass, and blood. Dissected tissue that was presumed to be a lymph node was verified as a lymph node by fixing the tissue in formalin for later histological sectioning and analysis.
[0028] Radioactivity was monitored using two Neoprobes (Model 1000, Neoprobe Corp., Dublin, Ohio). The readings were converted to counts per minute (CPM). Three readings were taken for each rabbit at each time point, and were averaged. The average for 9 three rabbits at each time point was calculated for each time period. Background levels were determined over the base of the neck.
[0029] The radioactivity measured in the right paw (the. site of injection) remained steady for the entire 48 hr, with mean levels from 1.6 million CPM at 15 min to 1.4 million CPM at 48 hr. (Fig. 1) For the lymph node readings, in vivo and in vitro readings were quite similar (Fig. 2 and Fig. 3, respectively), with the highest readings at 15 min post-injection (an average of 1 million CPM). The readings slowly declined over the 48 hr to about 100,000 CPM. The background levels in the neck (Fig. 4), as well as in the left paw (Fig. 5) and blood (Fig; 6), were always less than 100,000 CPM, with the lowest reading at 48 hr. The decline over the 48 hr period represented both decay and elimination of radioactivity.
[0030] Among the animals examined for biodistribution, no selective uptake was identified in the 'stomach, spleen, liver, ovary or testes, heart, lungs, or thyroid. (Fig. 6) However, a gradual increase was seen in the kidney from 2 hr to 8 hr, and then a fall at 24 hr and 48 hr. (Fig. 7). The right groin levels increased over time until 8 hr, then declined to 60,000 CPM at 48 hr. (Fig. 8) The right groin measurements indicated that the radioactive dye passed through the knee lymph node (the popliteal) into the right groin.
[0031] These results confirmed that the radiolabeled dye rapidly went to the popliteal nodes. The tissue that was blue and radioactive was nodal tissue, as confirmed by histology. (Data not shown) The radioactivity reached the node within 15 min. This is much quicker than has been reported for 99mTc sulfur colloid, which takes from 30 min to an hour to travel to the lymph node.
EXAMPLE 2 Additional Animal Model Confirming Identification of Sentinel Lymph Node Using the One-Step Procedure
[0032] To evaluate the use of the one-step procedure in a second animal model, mini-pigs · (of about 75 lb) will be used. These animals are used in a training course for identification of sentinel lymph nodes offered by the Endo-Surgery Institute (Cincinnati, Ohio). The pigs will be anesthetized and then injected with a 5 cc mixture similar to that used in rabbits in Example 1. The injection will be a subcutaneous injection of the anterior thigh. After 10 min, the inguinal and groin regions of the pig will be scanned with a handheld probe to determine regions with radioactivity ("hot" regions). An incision will be made near the hot regions to look for blue areas and discern the source of the radioactivity. Any node that is either "hot" or blue will be extracted. The location of each node will be noted. Nodal tissue will be confirmed by histochemistry.
[0033] The sentinel lymph nodes will be identified as follows: (1) Nodes that are stained blue; (2) Nodes with an afferent lymphatic channel that is stained blue; and (3) All nodes with a radioactive count that is greater than 10% of that of the sentinel lymph node having the highest count (using a hand-held gamma detector (e.g., Neoprobe; or C-Trak™, Care Wise Medical Products, Morgan Hill, California). All sentinel nodes identified by the criteria listed above will be removed and stained with hematoxylin, eosin, and cytokeratin to corifirm that the tissue is lymph nodal tissue. For each removed node, the presence or absence of dye and radioactivity, both in vivo and ex vivo, will be recorded.
[0034] We expect these results to confirm that the radiolabeled dye rapidly goes to the major, sentinel lymph nodes, and that this technique accurately identifies the sentinel lymph node(s). It is also expected that radioactivity will reach the node within 15 min, a much quicker time than has been reported for 99mTc sulfur, colloid, which takes from 30 min to an hour to travel to the lymph node.
EXAMPLE 3 Clinical Studies of the One-Step Procedure
[0035] To evaluate the use of the one-step procedure in human patients, patients with i breast cancer will be recruited under the guidelines and approval of the local Institutional Review Board. Patients will be selected who have breast cancer in either Stage I or II, and whose axillary node status is either NO or Nl . Patients will be excluded who are pregnant or nursing or who have a known sensitivity to iodine, isosulfan, lymphazurin, or methylene blue dye.
[0036] The patients will be divided into two groups, to be injected at either a peritumoral or subareolar site. Each group will be injected with a 5 cc mixture of radioactive methylene blue (0.5 cc I25I-methylene blue) and unlabeled blue dye (4.5 cc lymphazurin or 11 methylene blue). Distribution of the injected material will be aided by either breast massage or breast compression for about 5 to about 8 min after injection.
[0037] The sentinel lymph nodes will be identified as follows: (1) Nodes that are stained blue; (2) Nodes with an afferent lymphatic channel that is stained blue; and (3) All nodes with a radioactive count that is greater than 10% of that of the sentinel lymph node having the highest count (using a hand-held gamma detector (e.g., Nedprobe; or C-Trak™, Care Wise Medical Products, Morgan Hill, California). All sentinel nodes identified by/the criteria listed above will be removed and stained with hematoxylin, eosin, and cytokeratin to determine the presence of malignant cells. For each removed node, the presence or absence of dye and radioactivity, both in vivo and ex vivo, will be recorded.
[0038] I expect this one-step procedure will be as effective as the two-step, dual mapping procedure that is currently widely used. I expect the radiolabeled dye to travel to the lymph nodes faster than would the larger 99mTc sulfur colloid, and thus to be more useful as during surgery.
EXAMPLE 4 Human Clinical Trials of the One-Step Procedure
[0039] Radiolabeled methylene blue. A prospective Phase I/II trial of 125I methylene blue for< the intraoperative detection of sentinel nodes in twelve women with invasive breast cancer was conducted. Methylene blue (1% USP) used in the protocol was obtained commercially as a sterile pyrogen-free product (Faulding Pharmaceutical Co., Paramus New Jersey). This product was labeled with 125Iodine (125I) using a proprietary iodogen method, purified, and its sterility and pyrogeniciry determined prior to distribution. The product was commercially obtained from Iso-Tex, Friendswood Texas. Purity of the drug was confirmed using mass spectrometry of 127I- labeled (by identical parallel reactions) product and HPLC. Each batch was tested for radiostability. The final product has a shelf life of 120 days.
[0040] The decay-corrected radioactive content of the final admixture of cold and hot products (125I methylene blue) ranged from 100 u,Ci/5cc to 1000 μΟ/5οο. The physical half-life of 125I is 60 days, however in preliminary animal experiments the biologic half-life of the 12 radiolabeled dye was about 6 hours (Data not shown). This correlated well with the known plasma half-life of non-labeled methylene blue (5.4 hours).
[0041] Patients: Twelve women with pathologically proven invasive breast cancer were enrolled in this trial between December 2004 and May 2005. Clearance to use the experimental drug 125I Methylene Blue in this Phase Ι/ΙΓ pilot trial was granted following submission of an Investigational New Drug (IND #70627) application through the FDA and approval by the 1KB for the Louisiana State University Health Sciences Center, New Orleans, Louisiana. Participating surgeons in this trial were considered to be experts in sentinel lymph node biopsy techniques for breast cancer. In general, the surgical techniques used for sentinel node biopsy in this series were based on the National Surgical Adjuvant Breast and Bowel Program J(NSABP) guidelines. The inclusion criteria for tins study included female subjects with Stage 0, I, or Π invasive breast cancer with a clinical axillary node status of NO. The exclusion criteria for this study declined subjects known to be pregnant or nursing, incarcerated prisoners, subjects under the age of 18 years, and subjects with a known allergy to shellfish, iodine or methylene blue dye.
[0042] ^ Preoperative Patient Preparation. To reduce the potential for 125I uptake in the thyroid, all patients were given ten drops a day of a saturated iodine solution (Lugol's solution) orally for two days prior to surgery, the day of surgery, and for three days thereafter.
[0043] Intraoperative Techniques. Following induction of general anesthesia, patients were injected with a combination of unlabeled methylene blue and I25I methylene blue in doses ranging from 100 microcuries to 1000 μθϊ. The total number of patients was twelve. ;Two patients were given each dose combination; i.e., two patients received either 100, 200, 300, 400, 500, or 1000 μθί of 125I methylene blue dye combined with unlabeled t methylene blue dye in a total volume of 5 cc. Patients were injected four times in the peritumoral or subareolar location using 1.25 c.c aliquots in the 3-, 6-, 9-, and 12-o'clock position of breast tissue surrounding the tumor or areola. Decisions regarding peritumoral or subareolar injection were at the discretion of the operating surgeon. Counting of the radioactive emissions at the primary injection site [counts per second (cps) averaged over ten seconds] ' was performed and recorded immediately following injection. Manual massage and ! 13 compression of the injected breast was then performed for 10 minutes following injection. A hand-held gamma detector (Neoprobe, Model 1000, Dublin Ohio) was used to scan the axilla once each minute beginning at 15 minutes post-injection. Transcutaneous 'hot spots' in the axilla were defined as radioactive counts (cps) consistently higher than the adjacent background. Failure to elicit a significant 'hot spot' in the axilla within 20 minutes prompted the surgeon to perform 'flushing' of the primary injection site with 25-50 cc of sterile NaCl solution per the NSABP B-32 protocol training manual. The time interval necessary for discovery of the 'hot spot' following primary injection was also recorded. An incision was made in the axilla overlying the 'hot spot'. The hand-held gamma detector was placed within the wound to facilitate a more precise detection of the sentinel lymph node. Lymph nodes that were significantly higher in radioactive content than adjacent axillary tissue were considered 'hot' sentinel lymph nodes and excised. In vivo and ex vivo nodal counts and axillary background counts were performed and recorded before and following removal of the sentinel node. Lymph nodes with in vivo counts greater than 10% of the in vivo counts obtained with the first node identified were also considered sentinel lymph nodes. Lymph nodes that were stained blue and/or having afferent lymphatic channels stained blue were also considered to be sentinel lymph nodes and excised. In addition, lymph nodes that contained both significant radioactive counts and were stained blue were categorized as 'hot & blue' sentinel lymph nodes. All sentinel lymph nodes identified by the established criteria were labeled, submitted individually, and delivered to pathology where they were stained with hematoxylin and eosin and for the presence of cytokeratin (immunohistochemistry) following serial step sectioning. All information regarding separate lymph nodes was reported individually.
[0044] Postoperative procedure. All patients received standard postoperative care, and continued to take oral Lugol's solution on the day of surgery and for three days thereafter. Patients were scanned postoperatively at 7 and 14 days with a hand-held gamma detector for detection of residual radioactivity in the primary injection site, the axilla, and the thyroid gland.
[0045] Statistical Analysis. Doses of 125I methylene blue varied from 100 to 1000 μθϊ. The effect of increasing dose on the injection site count rate, the in vivo count rate 14 for sentinel node and the ex vivo count rate were analyzed by regression analysis. Descriptive statistics (mean ± SD) were used to describe differences in demographic information and other differences between variables.
[0046] Patient demographics. The mean age (±SD) of the twelve women was 51 ± 8 years. Four of the women were Caucasian, seven were African-American, and one was Hispanic. All patients were originally diagnosed with invasive breast cancer. Eleven patients underwent needle core biopsy, and one underwent open incisional biopsy to establish a tissue diagnosis. All patients were staged clinically as Stage I or II prior to surgery. Two patients underwent neoadjuvant chemotherapy prior to definitive surgery. All of the patients initially underwent lumpectomy and sentinel lymph node biopsy. Three of the patients were found to have metastatic disease in the sentinel nodes and underwent subsequent axillary dissection.
[0047] Identification of Sentinel Lymph Nodes. Four of the patients received peritumoral injection and eight received subareolar injection. The mean for primary injection site counts (±SD) was 24,050 ± 11,664 and ranged from 3,346 to 47,300 (Raw data not shown). The cps at the primary injection site correlated well with the dose injected (R2 = p < .001). All patients were injected with the full 5cc volume of l25I methylene blue solution. Six patients received a supplemental 25cc sterile NaCl solution 'flush' injection to facilitate lymphatic drainage according to NSABP guidelines. Sentinel nodes were detected in 11 of 12 patients. The mean number of nodes per patient detected was 1.66. The mean time interval (±SD) between time of injection and transcutaneous detection of the sentinel lymph node was 22 ± 2.45 minutes. Hot spots in the axilla were transcutaneously identified in 11 of 12 patients. All hot spots involved nodes in the Level I axillary node group. A sentinel node was found underneath the dermal hot spot in all instances. Of the 19 total sentinel lymph nodes discovered, 15 nodes were classified as 'hot & blue' and 2 nodes as 'blue only'. No nodes met the criteria for 'hot only'. The mean in vivo counts (+/-SD) for all 'hot & blue' nodes was 391 i 324 and ranged from 20 to 1,228; however, the mean in vivo (iSD) counts for patients receiving doses ranging from 400 μθϊ to 1,000 μθϊ was 531 ± 289, with values ranging from 55 to 1,228. The mean ex vivo counts (± SD) for all 'hot & blue' nodes was 264 ± 347 and ranged from 14 to 1,516; however, the ex vivo counts (± SD) for patients receiving doses ranging from 400 μθϊ to 1,000 μθί (n=6), was 310 ± 419, with values ranging from 25 to 1,516. The mean axilla background counts were 19 ± 29 and ranged from 0 to 107cps.
[0048] No adverse local, systemic or anaphylactic reactions were noted during or following injection of the 125I methylene blue. No evidence of skin necrosis in the vicinity of the primary injection site was observed. One patient receiving a 200 μθί dose developed a local infection in the lumpectomy wound that resolved with local wound care. One patient receiving a dose of 300 μθϊ developed a seroma one week postoperatively. The radioactive count at the breast wound site was recorded as 3,100 cps at that time. Drainage of the seroma resulted in dramatic decrease in wound counts (77 cps) one week later.
[0049] The mean radioactive counts (±SD) of the thyroid gland, axillary wound, breast lumpectomy wound, and background radioactive counts one week postoperatively were 26 ± 7, 22 ± 12, 349 ± 833 and 4 ± 2cps, respectively.
[0050] The maximum dose utilized in this study for 125I Methylene Blue (1000 μθΐ) represented one tenth the radioactivity injected in the maximum recommended, and typically used, dose of 10 mCi for 99mTc sulphur colloid. Without wishing to be bound by this theory, it is believed that doses up to 2000 μθϊ could be used. The dose effective in this procedure would be from about 300 to about 2000 μθϊ, more preferably from about 500 to 1500 μθϊ, and most preferably about 1000 μθϊ. Obviously less radioactive exposure provides a higher level of safety for patients, surgeons, and perioperative personnel. In addition, the energy of 99mTc is 140 Kev as compared to 35 Kev from 125I.
[0051] 125I methylene blue as a single entity was successfully injected intraoperatively and the radiolabeled dye quickly transited through the lymphatics to the sentinel node groups. In this study at doses of 400 μθϊ and higher, transcutaneous detection of 'hot spots' was achieved approximately 20 minutes post-injection. At lower doses (100-200 μθί) trancutaneous detection of 'hot spots' was achieved within the same time period, but only following a 25cc sterile NaCl 'flushing' at the primary injection site to facilitate lymphatic drainage. Flushing procedures were used as an adjunct in six subjects. Regardless of the administered dose, nine of ten patients demonstrated transit of detectable radioactivity to the sentinel lymph nodes. The remaining subject had no sentinel lymph nodes detected by 16 radiotracer or by the blue dye. Based on these results it appears that conjugated dye and radiotracer effectively transit from the primary injection site to the sentinel lymph nodes. The lower emitted energy of 125I gamma particles necessitates an injected dose of at least 300 μθί to allow reliable transcutaneous detection, but injections of 1000 μθ were proven to be more effective. No significant difference in time interval for initial detection of 'hot spot' or eventual detection of sentinel node was noted between the patients undergoing peritumoral versus subareolar injection. In general, patients with upper lateral quadrant tumors were given subareolar injections. No adverse reactions were attributed to injection of 125I methylene blue or unlabelled methylene blue dye. Superficial injections administered in a subareolar location exhibited no incidence of skin necrosis.
[0052] The use of l25I methylene blue for labeling of sentinel lymph nodes in the axilla may prove to be an effective means of reliable detection of sentinel lymph nodes in selected breast cancer patients. Administration of the drug was proven to be safe for humans with no incidence of adverse reactions or residual radioactive uptake by the thyroid gland within this study. An effective dose of 1000 μθϊ was identified that allows for reliable transcutaneous detection of the sentinel lymph node. This technique eliminates painful preoperative injections of 99mTc colloid, is performed by the surgeon in the operating room, is associated with lower radiation exposures for personnel and avoids the delays caused by non-operating room personnel.
[0053] The complete disclosures of all references cited in this specification are hereby incorporated by reference. In the event of an otherwise irreconcilable conflict, however, the present specification shall control. 17

Claims (10)

1. 80635/3 1. Use of radiolabeled methylene blue in the manufacture of an injectable medicament for identifying,; in a mammalian patient with a solid tumor, one or more sentinel lymph nodes to which lymph from the tumor initially flows; wherein said injectable medicament is suitable for a method comprising the steps of: (a) injecting radiolabeled methylene blue in or near the tumor or tumor site; (b) identifying as a sentinel lymph node a lymph node that, within about 20 minutes following said injecting, has accumulated a detectable fraction of the radiolabeled methylene blue, as indicated optically by the presence of the dye methylene blue, or as indicated radiologically by decay products of the radiolabel, or both; and (c) surgically removing the identified one or more sentinel lymph nodes.
2. A use as recited in claim 1, wherein said identifying step comprises identifying the location of one or more sentinel lymph nodes radiologically during surgery; and then further identifying the location of the one or more sentinel lymph nodes by optically locating dyed tissue during surgery, in one or more locations that had thus been indicated radiologically; and wherein said method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
3. A use as recited in claim 1, wherein the radiolabled methylene blue is present in said injectable medicament as an admixture with a non-radiolabeled dye; wherein the presence of the non-radiolabeled dye in a tissue may be observed optically due to the non-radiolabeled dye's visible, ultraviolet, or infrared absorption, fluorescence, luminescence, or phosphorescence; wherein the molecular weight and chemical properties of the non-radiolabeled dye are such that a detectable fraction of the non-radiolabeled dye will, following injection, travel from the tumor or the vicinity of the tumor to one or more sentinel lymph nodes within about 20 minutes.
4. A use as recited in claim 3, wherein said identifying step comprises identifying the location of one or more sentinel lymph nodes radiologically during surgery; and wherein said method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
5. A use as recited in claim 3, wherein said identifying step comprises identifying the location of one or more sentinel lymph nodes by optically locating dyed tissue during surgery; and wherein said method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
6. A use as recited in claim 3, wherein said identifying step comprises identifying the location of one or more sentinel lymph nodes radiologically during surgery; and then further identifying the location of the one or more sentinel lymph nodes by optically locating dyed tissue during surgery, in one or more locations that had thus been indicated radiologically; and wherein said method additionally comprises the step, following said identifying, of surgically removing the identified one or more sentinel lymph nodes.
7. A use as recited in claim 3, wherein the non-radiolabeled dye is methylene blue.
8. A use as recited in claim 3, wherein the ratio of the non-radiolabeled dye to the radiolabled methylene blue is at least about 10: 1.
9. The use of claim 3, wherein the molecular weight and chemical properties of the non-radiolabeled dye are such that a detectable fraction of the dye will, following injection, travel from the injection site to one or more sentinel lymph nodes within about 15 minutes.
10. The use of claim 3, wherein the molecular weight and chemical properties of the non-radiolabeled dye are such that a detectable fraction of the dye will, following injection, travel from the injection site to one or more sentinel lymph nodes within about 10 minutes. 18 180635/3 1 1. The use of claim 1, wherein the radiolabled methylene blue is I-methylene blue. 12. The use of claim 3, wherein the radiolabled methylene blue is 125I-methylene blue and the non-radiolabeled dye is isosulfan blue. 13. The use of claim 1, wherein the tumor is selected from the group consisting of melanoma, breast cancer, head and neck cancer, lung cancer, bladder cancer, neuroendocrine cancer, squamous carcinoma, prostate cancer, gastric cancer, cervical cancer, vulvar cancer, thyroid cancer, pancreatic cancer, renal cancer, esophageal cancer, rectal cancer, penile cancer, lymphoma, multiple myloma, Merkel cell tumors, ovarian cancer, and colorectal cancer. 14. The use of claim 1, wherein the tumor is breast cancer. 15. The use of claim 1, wherein the tumor is melanoma. 16. The use of claim 1 wherein the radiolabel emits particles with an energy less than about 300 keV. 17. The use of claim 1 wherein the radiolabel emits particles with an energy less than about 150 keV. 18. The use of claim 1 wherein the radiolabel emits particles with an energy less than about 50 keV. 19. The use of claim 1 , wherein the radiolabel is an isotope of an element selected from the group consisting of Tc, Re, Mn, Fe, Se, Co, Ni, Zn, Cd, Mo, W, Cu, Ag, Au, Tl, Hg, Cr, Rh, B, I, CI, F, At, Y, Lu, Gd, Ga, Ho, In, Sm and Yb. 21. The use of claim 1 , wherein the radiolabel is I. 22. The use of claim 3, wherein the non-radiolabeled dye is selected from the group consisting of patent blue dye, isosulfan blue, methylene blue, fluorescein, tartrazine, iodocyanine green, rose Bengal, and congo red. For the Applicant, LOUI 90051/1.4 19
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